Jesus A Cabrera

Role of Store-Operated Calcium Channels and Calcium Sensitization in Normoxic Contraction of the Ductus Arteriosus

Background— At birth, the increase in oxygen causes contraction of the ductus arteriosus, thus diverting blood flow to the lungs. Although this contraction is modulated by substances such as endothelin and dilator prostaglandins, normoxic contraction is an intrinsic property of ductus smooth muscle. Normoxic inhibition of potassium channels causes membrane depolarization and calcium entry through L-type calcium channels. However, the studies reported here show that after inhibition of this pathway there is still substantial normoxic contraction, indicating the involvement of additional mechanisms. Methods and Results— Using ductus ring experiments, calcium imaging, reverse-transcription polymerase chain reaction, Western blot, and cellular electrophysiology, we find that this depolarization-independent contraction is caused by release of calcium from the IP3-sensitive store in the sarcoplasmic reticulum, by subsequent calcium entry through store-operated channels, and by increased calcium sensitization of actin-myosin filaments, involving Rho-kinase. Conclusions— Much of the normoxic contraction of the ductus arteriosus at birth is related to calcium entry through store-operated channels, encoded by the transient receptor potential superfamily of genes, and to increased calcium sensitization. A clearer understanding of the mechanisms involved in normoxic contraction of the ductus will permit the development of better therapy to close the patent ductus arteriosus, which constitutes ≈10% of all congenital heart disease and is especially common in premature infants.

Generation of Oxidants by Hypoxic Human Pulmonary and Coronary Smooth-Muscle Cells

BACKGROUND Pulmonary vasoconstriction in response to hypoxia is unusual inasmuch as local exposure of nonpulmonary vasculature to hypoxia results in vasodilation. It has been suggested that pulmonary artery smooth-muscle cells may relax in response to intracellular generation of reactive oxygen species (ROS) and that the production of ROS decreases under hypoxia. However, other workers report increased ROS production in human pulmonary artery smooth-muscle cells (HPASMC) during hypoxia. METHODS Using dihydrodichlorofluorescein diacetate, dihydroethidium, and Amplex Red (Molecular Probes; Eugene, OR), we estimated ROS generation by confluent primary cultures of HPASMC and human coronary artery smooth-muscle cells (HCASMC) under normoxia (20%) and acute hypoxia (5%). RESULTS All three assay systems showed that HPASMC production of ROS is decreased under hypoxia and to a greater extent than the decrease in ROS production by HCASMC. A substantially greater percentage of normoxic ROS production by HPASMC is mitochondrial (> 60%) compared to HCASMC (< 30%). CONCLUSIONS These results support the conclusion that ROS generation decreases, rather than increases, in HPASMC during hypoxia. However, as ROS production also decreases in HCASMC during hypoxia, the reason for the opposite change in vascular tone is not yet apparent.

Continued depression of maximal oxygen consumption and mitochondrial proteomic expression despite successful coronary artery bypass grafting in a swine model of hibernation

OBJECTIVE Clinical studies indicate incomplete functional recovery of hibernating myocardium after coronary artery bypass grafting. We hypothesized that persistent contractile abnormalities after coronary artery bypass grafting are associated with decreased mitochondrial proteins involving electron transport chain that might limit maximal oxygen consumption. METHODS Seven pigs with hibernating myocardium underwent off-pump revascularization with left internal thoracic artery to mid left anterior descending artery. At 4 weeks, left internal thoracic artery anastomosis was patent by multidetector computed tomography. Regional function (transthoracic echocardiography) and blood flow (microspheres) were assessed at rest and during high-dose dobutamine (40 μg/[kg · min]). Expression of electron transport chain proteins was analyzed with isobaric tags for relative and absolute quantification. RESULTS After revascularization, multidetector computed tomography confirmed severe left anterior descending stenosis and patent left internal thoracic artery graft. Regional function and blood flow normalized at rest; however, function in left anterior descending distribution remained depressed relative to remote regions, and myocardial blood flow in that region did not increase normally when challenged with high-work state. Concomitant with reduced maximal blood flow response in left anterior descending region was more than 40% reduction in electron transport chain proteins essential to adenosine triphosphate production. CONCLUSIONS Despite successful revascularization of hibernating myocardium, regional function and blood flow remained depressed during catecholamine stress. Electron transport chain proteins known to be downregulated during adaptive process within hibernating myocardium did not normalize after revascularization. These data demonstrate a potential bioenergetic cause of persistent dysfunction and heart failure within successfully revascularized hibernating myocardium.

Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

Uncoupling Protein-2 Expression and Effects on Mitochondrial Membrane Potential and Oxidant Stress in Heart Tissue

Myocardial uncoupling protein (UCP)-2 is increased with chronic peroxisome proliferator-activated receptor γ (PPARγ) stimulation, but the effect on membrane potential and superoxide is unclear. Wild-type (WT) and UCP-2 knockout (KO) mice were given a 3-week diet of control (C) or the PPARγ agonist pioglitazone (PIO; 50 μg/g-chow per day). In isolated mitochondria, UCP-2 content by Western blots, membrane potential (ΔΨm) by tetraphenylphosphonium (TPP), and relative superoxide levels by dihydroethidium (DHE) were measured. Oxygen respiration was determined at baseline and after 10 min anoxia-reoxygenation. PIO induced a 2-fold increase in UCP-2 and nuclear-bound PGC1α in WT mice with no UCP-2 expression in KO mice. Mitochondrial ΔΨm from WT mice on C and PIO diets was -166±4 mV and -147±6 mV, respectively (P<0.05). These values were lower than in UCP-2 KO mice on C and PIO (-180±4 mV and -180±4 mV, respectively; P<0.05). Maximal complex III inhibitable superoxide from WT mice on C and PIO diets was 22.5±1.3 and 17.8±1.1 AU, respectively (P<0.05), and were lower than UCP-2 KO on C and PIO (32.9±2.3 and 29.2±1.9 AU, respectively; P<0.05). Postanoxia, the respiratory control index (RCI) in mitochondria from WT mice with and without PIO was 2.5±0.3 and 2.4±0.2, respectively, and exceeded that of UCP-2 KO mice on C and PIO (1.2±0.1 and 1.4±0.1, respectively; P<0.05). In summary, chronic PPARγ stimulation leads to depolarization of the inner membrane and reduced superoxide of isolated heart mitochondria, which was critically dependent on increased expression of UCP-2. Thus, UCP-2 expression affords resistance to brief anoxia-reoxygenation.

Mechanisms of oxygen sensing : a key to therapy of pulmonary hypertension and patent ductus arteriosus

Specialized tissues that sense acute changes in the local oxygen tension include type 1 cells of the carotid body, neuroepithelial bodies in the lungs, and smooth muscle cells of the resistance pulmonary arteries and the ductus arteriosus (DA). Hypoxia inhibits outward potassium current in carotid body type 1 cells, leading to depolarization and calcium entry through L‐type calcium channels. Increased intracellular calcium concentration ([Ca++]i) leads to exocytosis of neurotransmitters, thus stimulating the carotid sinus nerve and respiration. The same K+ channel inhibition occurs with hypoxia in pulmonary artery smooth muscle cells (PASMCs), causing contraction and providing part of the mechanism of hypoxic pulmonary vasoconstriction (HPV). In the SMCs of the DA, the mechanism works in reverse. It is the shift from hypoxia to normoxia that inhibits K+ channels and causes normoxic ductal contraction. In both PA and DA, the contraction is augmented by release of Ca++ from the sarcoplasmic reticulum, entry of Ca++ through store‐operated channels (SOC) and by Ca++ sensitization. The same three ‘executive’ mechanisms are partly responsible for idiopathic pulmonary arterial hypertension (IPAH). While vasoconstrictor mediators constrict both PA and DA and vasodilators dilate both vessels, only redox changes mimic oxygen by having directly opposite effects on the K+ channels, membrane potential, [Ca++]i and tone in the PA and DA. There are several different hypotheses as to how redox might alter tone, which remain to be resolved. However, understanding the mechanism will facilitate drug development for pulmonary hypertension and patent DA.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12 weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2 min, each separated by 2 min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport.

The role of ion channels in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which localized flow of blood in small resistance pulmonary arteries is matched to alveolar ventilation. This chapter discusses the role of several potassium and calcium channels in HPV, both in enhancing calcium influx into smooth muscle cells (SMCs) and in stimulating the release of calcium from the sarcoplasmic reticulum, thus increasing cytosolic calcium. The increase in calcium sensitivity caused by hypoxia is reviewed in Chapter 19. Particular attention is paid to the activity of the L-type calcium channels which increase calcium influx as a result of membrane depolarization and also increase calcium influx at any given membrane potential in response to hypoxia. In addition, activation of the L-type calcium channel may, in the absence of any calcium influx, cause calcium release from the sarcoplasmic reticulum. Many of these mechanisms have been reported to be involved in both HPV and in normoxic contraction of the ductus arteriosus.